Ester compound and use thereof

ABSTRACT

It is an object of the present invention to provide a novel ester compound and the use thereof as a sex attractant. The present invention provides a compound represented by formula (I) described below, and a sex attractant comprising, as an active ingredient, this compound:

TECHNICAL FIELD

The present invention relates to a novel ester compound and a sexattractant containing the above compound as an active ingredient.

BACKGROUND ART

Pseudococcus cryptus is the most important insect pest growing inJapanese citrus fruits, which causes enormous damage to greenhouseculture such as the culture of hothouse oranges. Pseudococcus cryptushas a strong characteristic that it hides at an indistinctive site suchas a space between leaves, and thus, it is difficult to find thepresence thereof when the insect pests exist at a low density. Since thecontrol of these insect pests is carried out after the pests have causedcertain damage, the control may be too late to start in many cases.Thus, it is an urgent issue to efficiently forecast of emergence of theinsect pests.

In general, many scale insects including Pseudococcidae have the form ofan adult insect that is significant different between males and females.An adult male has wings for flying, but its life time is extremelyshort, such as a duration of life between several hours to 1 day. Incontrast, an adult female does not have such wings and thus does nothave high migration ability, but it has a longer life time than an adultmale. Accordingly, an adult male should find an adult female to mate itwithin a limited period of time. In order to efficiently find females,some scale insects utilize their sex pheromone. In recent years,identification of pheromones of scale insects has progressed, and thestructures and components of pheromones of several scale insects havebeen identified (e.g. Planococcus citri, Pseudococcus comstocki,Planococcus ficus, Aonidiella aurantii, Aonidiella citrina,Comstockaspis perniciosa, Pseudaulacaspis pentagona, 5 types belongingto Matsucoccus, etc. (please refer to Non-Patent Documents 1 to 12)).

On the other hand, studies regarding agents or techniques forcontrolling insects utilizing sex pheromones of the insects, such asmass trapping or communication disruption between males and females,have actively been conducted. In these methods, using sex pheromone,adult males are attracted to a certain place, and the attracted insectsare then captured and killed. Otherwise, a normal mating action betweenmales and females is artificially disturbed to reduce the population ofthe next generation, thereby controlling insect pests. In addition, itis also possible to forecast the development of insect pests that aretargets of control, using such sex pheromone. It is considered that thecontrol of insect pests using sex pheromone is an eco-friendlytechnique, which reduces burden on the environment. It is anticipatedthat such a technique will be developed for more types of insect pestsin future.

Under such circumstances, as a result of studies regarding the ecologyof Pseudococcus cryptus and its natural enemy, the present inventorshave found for the first time that Pseudococcus cryptus also has sexpheromone.

[Patent Document 1]

JP Patent Publication (Kokoku) No. 61-036738 B (1986)

[Non-Patent Document 1]

Bierl-Leonhardt B. A., D. S. Moreno, M. Schwarz, H. S. Forster, J. R.Plimmer and E. D. DeVilbiss (1980) Identification of the pheromone ofthe comstock mealybug. Life Science 27: 399–402.

[Non-Patent Document 2]

Bierl-Leonhardt B. A., D. S. Moreno, M. Schwarz, J. Fargerlund and J. R.Plimmer (1981) Isolation, identification and synthesis of the sexpheromone of the citrus mealybug, Planococcus citri (Risso). Tetrahedr.Lett. 22: 389–392.

[Non-Patent Document 3]

Dunkelblum E., Z. Mendel, F. Assael, M. Harel, L. Kerhoas and J. Einhorn(1993) Identification of the female sex pheromone of the Israeli pinebast scale Matsucoccus josephi. Tetrahedr. Lett. 34:2805–2808.

[Non-Patent Document 4]

Einhorn J., P. Menassieu, C. Malosse and P. Ducrot (1990) Identificationof the sex pheromone of the maritime pine scale Matsucoccus feytaudi.Tetrahedr. Lett. 31: 6633–6636.

[Non-Patent Document 5]

Gieselmann M. J., D. S. Moreno, J. Fargerlund, H. Tashiro and W. L.Roelofs (1979a) Identification of the sex pheromone of the yellow scale.J. Chem. Ecol. 5: 27–33.

[Non-Patent Document 6]

Gieselmann M. J., R. E. Rice, R. A. Jones and W. L. Roelofs (1979b) Sexpheromone of the San Jose scale. J. Chem. Ecol. 5:891–900.

[Non-Patent Document 7]

Heath R. R., J. R. McLaughlin, J. H. Tumlinson, T. R. Ashley and R. E.Doolittle (1979) Identification of the white peach scale sex pheromone:An illustration of micro techniques. J. Chem. Ecol. 5:941–953.

[Non-Patent Document 8]

Hinkens D. M., J. S. McElfresh and J. G. Millar (2001) Identificationand synthesis of the sex pheromone of the vine mealybug, Planococcusficus. Tetrahedr. Lett. 42: 1619–1621.

[Non-Patent Document 9]

Lanier G. N., Y. Qi, J. R. West, S. C. Park, F. X. Webstar and R. M.Silverstein (1989) Identification of the sex pheromone of threeMatsucoccus pine bast scales. J. Chem. Ecol. 15: 1645–1659.

[Non-Patent Document 10]

Negishi T., M. Uchida, Y. Tamaki, K. Mori, T. Ishiwatari, S. Asano andK. Nakagawa (1980) Sex pheromone of the comstock mealybug, Pseudococcuscomstocki Kuwana: Isolation and identification. Appl. Entomol. Zool. 15:328–333.

[Non-Patent Document 11]

Roelofs W., M. Gieselmann, A. Carde, H. Tashiro, D. S. Moreno, C. A.Henrick and R. J. Anderson (1977) Sex pheromone of the California redscale, Aonidiella aurantii. Nature 267: 698–699.

[Non-Patent Document 12]

Roelofs W., M. Gieselmann, A. Carde, H. Tashiro, D. S. Moreno, C. A.Henrick and R. J. Anderson (1978) Identification of the California redscale sex pheromone. J. Chem. Ecol. 4: 211–224.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a novel estercompound and a sex attractant containing the above compound as an activeingredient.

In order to achieve the aforementioned object under the currentcircumstances, the present inventors have conducted intensive studiesdirected towards searching for the sex pheromone of Pseudococcus cryptusbelonging to Pseudococcidae. As a result, they have succeeded inisolating the sex pheromone and determining the structure thereof. Thus,they have discovered the properties of the sex pheromone of Pseudococcuscryptus and the fact that the pheromone component is3-isopropenyl-2,2-dimethylcyclobutylmethyl 3-methyl-3-butenoate, therebycompleting the present invention.

That is to say, the present invention includes the following features:

-   (1) A compound represented by the following formula (I):

-   (2) A sex attractant comprising the compound represented by the    above-described formula (I) as an active ingredient.

The compound represented by the above-described formula (I) can beobtained by extraction and purification from Pseudococcus cryptus.

As an extracting solvent used herein, organic solvents may generally beused. Preferred examples may include: water-miscible organic solventssuch as methanol, ethanol, propanol, or acetone; and water-immiscibleorganic solvents such as ether, ethyl acetate, chloroform, pentane, orhexane. The obtained extract is concentrated and purified, so as toefficiently obtain an ester compound of interest.

Purification can be carried out by appropriately combining silica gelchromatography, reverse phase silica gel chromatography, adsorptionchromatography, reverse phase adsorption chromatography, gaschromatography, high performance liquid chromatography, reverse phasehigh performance liquid chromatography, and the like.

The thus obtained ester compound exhibits an action to sexually attractPseudococcus cryptus, and thus, it can be used as a sex attractant.

In order to use the compound of the present invention as a sexattractant, the above-described ester compound may directly be containedin a trap. However, in general, the above-described ester compound isdissolved in a suitable organic solvent such as pentane, hexane, diethylether, acetone, methylene chloride, or the like, and the obtainedproduct is then encapsulated in a rubber cap, a capillary, a plasticcapsule, or the like. Alternatively, the above product is carried andadsorbed on an activated carbon, an inactivated powder such as silicagel, or a granule, and it is then used. The amount and usage of thecompound of the present invention as a sex attractant are not limited.Generally, the compound of the present invention is contained in a sexattractant as prepared above at an order of ng. This sex attractant isplaced in a trap onto which an adhesive substance or the like has beenapplied. Thereafter, such a trap may be established for every 2 or 3trees in the orchard. Thus, adult males of Pseudococcus cryptus areattracted to the compound of the present invention and are eventuallycaptured by the trap onto which the adhesive substance has been applied.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a mass spectrum of an active ingredient of sex pheromone ofPseudococcus cryptus;

FIG. 2 shows a 600 MH_(z) 1H NMR spectrum of an active ingredient of sexpheromone of Pseudococcus cryptus; and

FIG. 3 is a view showing the configuration of hydrogen assigned by COSYanalysis and HOHAHA analysis.

The present specification includes part or all of the contents asdisclosed in the specification and/or drawings of JP Patent ApplicationNo. 2002-328482, which is a priority document of the presentapplication.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be more specifically described in thefollowing examples. However, the examples are not intended to limit thescope of the present invention.

EXAMPLE 1

-   (1) Isolation Operation    Extraction of Sex Pheromone

A pumpkin inoculated with 500 to 4,000 virgin females of Pseudococcuscryptus was placed in a 5-liter double-capped desiccator. Thereafter,pheromone accumulated in the desiccator was captured with 1 g of anadsorbent (Tenax GC). The period of time for capturing such pheromonewas set at approximately 30 days. The capturing time was set at 7 hoursper day, and the flow rate of the air was set at 7 liters per minute.The pheromone captured by the absorbent was extracted with 40 ml ofpentane at a rate of once every 3 days. In order to estimate the amountof pheromone captured, dead insects were removed every 3 or 4 days, andvirgin females were replenished. The collected extract (400 ml) wassubjected to vacuum concentration at room temperature, and the obtainedconcentrate (10 ml) was defined as a crude extract of sex pheromone ofPseudococcus cryptus.

Purification of Sex Pheromone

Subsequently, using the activity of attracting adult males ofPseudococcus cryptus as an indicator, sex pheromone was purified fromthe above-described crude extract. First, a sex pheromone crude extract(2 ml) obtained by subjecting vacuum concentration again wasfractionated by chromatography using a column filled with 30 g offlorisil (containing 7% water, 100–200 meshes, Floridin Co.). Elutionwas carried out using a mixed solution consisting of diethyl ether andhexane. Extraction was successively carried out using 50 ml of hexane,120 ml of 5% ether/hexane, 150 ml of 15% ether/hexane, 150 ml of 25%ether/hexane, and 150 ml of 50% ether/hexane. Thereafter, a 5%ether/hexane fraction (120 ml) exhibiting the highest attractingactivity was subjected to vacuum concentration. The thus concentratedfraction (10 ml) was purified by high performance liquid chromatography(hereinafter referred to as HPLC) (HEWLETT PACKARD SERIES 1050). InHPLC, a silica gel column (Inertsil, 5 μm, 4.6 mm I.D.×250 mm, GLScience Inc.) was used, and elution was carried out using a solvent witha composition of 3% ether/hexane at a flow rate of 1 ml/min. In HPLC, afraction (0.5 ml) with a retention time of 7.5 to 8 minutes exhibitedthe highest attracting activity. This fraction and the next fraction (afraction (0.5 ml) with a retention time of 8 to 8.5 minutes) weregathered, and the gathered fraction was then fractionated by gaschromatography (hereinafter referred to as GC). As a GC device, HEWLETTPACKARD 5890 SERIES II was used, and as a column, FFAP (0.56 mm I.D.×15m, a liquid phase thickness of 1 μm, GL Science Inc.) was used. Heliumwas used as a carrier gas, and the flow rate was set at 5 ml/min.Conditions for the temperature of an oven were set such that atemperature of 50° C. was retained for 1 minute and then the temperaturewas increased up to 220° C. at a rate of 5° C./min. In GC, a fractionwith a retention time of 20.5 to 21 minutes exhibited the highestattracting activity (please refer to Table 1 shown below). Theabove-described extraction and purification process was repeatedlycarried out, so as to obtain 3 ml of purified sex pheromone (500,000female-day equivalents/300 ml). In the present specification, the term“500,000 female-day equivalents” is used to mean the amount of pheromonereleased from 500,000 adult females per day.

TABLE 1 Activities of Florisil LC, HPLC, GC fractions of attractingadult males of Pseudococcus cryptus Rate of attracting adult males(%)^(a) Treated Significant Fraction group^(b) Control groupdifference^(c) Florisil Hexane  2.1 10.8 n.s.  5% E/H 76.5 0 ** 15% E/H54.4 2.1 * 25% E/H 56.7 4.1 n.s. 50% E/H 50.7 7.5 * HPLC    0–7 minutes20.1b 3.0 n.s.   7–10 minutes 80.5a 0 **   10–16 minutes 66.6a 37 *Blank 60.0a 0 ** HPLC 7–10 minutes  7–7.5 minutes 12.1b 4.2 n.s.  7.5–8minutes 74.8a 0 **  8–8.5 minutes 60.0ab 0 **  8.5–10 minutes 62.0ab 0** GC   0–15 minutes  3.0b 0 n.s.   15–18 minutes  2.6b 5.6 n.s. 18–19.5minutes  3.0b 0 n.s. 19.5–20 minutes 22.5b 0 ** 20–20.5 minutes  5.6b6.1 n.s. 20.5–21 minutes^(d) 79.6a 1.5 ** Blank 16.7b 0 n.s. ^(a)Thetotal value obtained by repeating the process 3 times ^(b)There are nosignificant differences among the same characters (Tukey-Kramer-test, p= 0.05). ^(c)The codes * and ** indicate that males were attracted tothe treated fraction group more strongly than to the control group atstandards of 5% and 1%, respectively. The code n.s. indicates that nodifferences were observed between both groups in terms of the attractingrate (t-test). ^(d)The total value obtained by repeating the process 5times

-   (2) Structure Determination    Analysis of Structure of Pheromone

The purified attracting active ingredient was analyzed with a gaschromatography-mass spectrometer (hereinafter referred to as GC-MS) interms of the molecular weight and molecular structure thereof (whereinan JEOL SX-102A double-focusing mass-spectrometer manufactured by JEOLwas used as an MS apparatus for GC-MS). HEWLETT PACKARD 5890 SERIES IIwas used as a GC apparatus for GC-MS, and FFAP (0.25 mm I.D.×30 m, aliquid phase thickness of 0.25 μm, GL Science Inc.) was used as acolumn. Helium was used as a carrier gas, and the flow rate was set at 1ml/min. Conditions for the temperature of an oven were set such that atemperature of 80° C. was retained for 1 minute and then the temperaturewas increased up to 210° C. at a rate of 7° C./min. An ionizationpotential was 70 eV, and an ionization current was 300 μA. Isobutane gaswas used as reaction gas in the measurement at a CI mode.

The isolated active ingredient had a retention time of 14.73 minutes inGC-MS. The active ingredient showed a peak with such characteristics asM/e: 69 (100%), 236 (8%), 168 (26%), 136 (22%), and 100 (32%) in an EImass spectrum. The mass number was estimated to be 236 from a CI massspectrum. In addition, as a result of high-resolution measurement, themass number was calculated to be 236.1808, and the elemental compositionwas estimated to be C₁₅H₂₄O₂. The retention time of a hydrogenationproduct of the isolated active ingredient was 13.35 minutes. Thisproduct showed a peak with such characteristics as M/e: 83 (100%), 170(25%), 143 (12%), 123 (10%), and 98 (69%) in an EI mass spectrum. Themass number of the hydrogenation product was estimated to be 240 from aCI mass spectrum, and it was assumed that it has two double bondstructures.

Analysis of Alcohol Moiety of Active Ingredient

Further, in order to determine the structure of an alcohol moiety of theisolated active ingredient, the active ingredient was subjected toalkaline hydrolysis and acetylation. 0.5 N KOH (2 ml) was added to aportion (0.1 ml) of the isolated active ingredient, and the mixture wasleft overnight. Thereafter, it was extracted with hexane (4 ml×3 times).Acetic anhydride (1 ml) was added to a hexane fraction (0.1 ml) obtainedby vacuum concentration, and the mixture was then left overnight.Thereafter, it was extracted with hexane (4 ml×3 times).

The hydrolysate of the isolated active ingredient was subjected to GC-MSanalysis. As a result, a substance having a retention time of 12.63minutes and the number of mass of 154 that was estimated from a CI massspectrum, was detected in neutral and basic fractions. This substanceshowed a peak with such characteristics as M/e: 71 (100%), 139 (12%),123 (9%), and 121 (27%) in an EI mass spectrum. Accordingly, it wasassumed that the active ingredient isolated by the above-describedpurification was an ester consisting of alcohol with a molecular weightof 154 and acid with a molecular weight of 100.

Subsequently, the alcohol moiety of the isolated active ingredient wasacetylated and then subjected to GC-MS analysis. As a result, theportion had a retention time of 10.63 minutes and showed a peak withsuch characteristics as M/e: 68 (100%), 196 (1.6%), 128 (20%), 121(19%), and 86 (9%) in an EI mass spectrum. The number of mass wasestimated to be 196 from a CI mass spectrum. The mass spectrum of thissubstance matched well with data regarding the sex pheromone ofPlanococcus citri. Moreover, it was found that this substance hadactivity of attracting adult males of Planococcus citri (please refer toTable 2 shown below). The level of such attracting activity was almostthe same as those of the crude extract of sex pheromone of Planococcuscitri and the acetylated alcohol moiety of sex pheromone of Planococcuscitri (please refer to Table 3 shown below). Thus, it was stronglysuggested that the acetylated alcohol moiety of the active ingredientisolated from Pseudococcus cryptus is a substance having a structureextremely similar to the sex pheromone of Planococcus citri, and thatthere is a possibility that the alcohol moiety of the active ingredientisolated from Pseudococcus cryptus is identical to the alcohol moiety ofthe sex pheromone of Planococcus citri.

Determination of Structure of Pheromone

The isolated active ingredient was analyzed by GC-MS. As a result, amass spectrum shown in FIG. 1 was obtained ([Mass Spectrum] Date: 19Jun. 2002 14:11; Data: 7362007; Sample: ryan; Note: Inlet: Direct; Ionmode: EI+; Spectrum Type: Normal Ion [MF-Linear]; RT: 0.87 min; Scan#:52,54; BP: m/z 69.0000; Int.: 53.66; Output m/z range: 34.6247 to290.4709; Cut Level: 0.00%; 594766). Moreover, the structure of theactive ingredient was analyzed by COSY and HOHAHA measurements using aproton NMR (JEOL A600; ¹H NMR 600 MH_(z); JEOL). As a result, an NMRspectrum shown in FIG. 2 was obtained ([STANDARD 1H OBSERVE] Date: 18Jun. 2002; Solvent: CDCl3; Ambient temperature; GEMINI-300 “varian”;PULSE SEQUENCE: Relax delay: 1.502 sec; Pulse 45.2 degrees Acq. time:3.498 sec; width 4500.5 Hz; 16 repetitions; OBSERVE: H1, 300.1350720MHz; DATA PROCESSING FT size 32768; Total time 1 minute). Hydrogen wasassigned as shown in FIG. 3. From these results, it was determined thatthe structure of sex pheromone with activity of attracting adult malesof Pseudococcus cryptus is represented by the following formula (I):

EXAMPLE 2

Sexual Attracting Activity

Biological Assay of Attracting Activity Using Petri Dish

Biological assay of sexually attracting activity shown in Tables 1 and 2was carried out as follows. First, two square filter papers with an areaof 1 cm² were placed on a Petri dish with a diameter of 90 mm.Thereafter, one paper was impregnated with each of sample solutions(shown in Tables 1 and 2) (100 μl of a stock solution in the case ofeach fraction shown in Table 1 and a crude extract shown in Table 2, and10 μl of a stock solution in the case of an acetylated product shown inTable 2), and it was defined as a treated group. The other paper wasimpregnated with only a solvent (100 μl), and it was defined as acontrol group. Hexane was used as such a solvent. The papers wereair-dried, and then, 10 to 20 adult males of Pseudococcus cryptus (inthe biological assay shown in Table 1) or Planococcus citri (in thebiological assay shown in Table 2) were released to the Petri dish. ThePetri dish was then capped. Approximately 10 to 60 minutes later, whenthe movement of the males stopped, the presence or absence of attractingactivity was confirmed from the ratio of males attracted to each filterpaper. The results are shown in the aforementioned table and Table 2.

TABLE 2 Activities of the sex pheromone of Planococcus citri and theacetylated substances of each sex pheromone of attracting adult males ofPlanococcus citri Rate of attracting adult males (%)^(a) SignificantAttracting source Treated group Control group difference^(b) Crudeextract of sex 52.8 0 ** pheromone of Planococcus citri Acetylatedproduct 1^(c) 48.9 0 ** Acetylated product 2^(d) 56.9 0 ** ^(a)The totalvalue obtained by repeating the process 3 times ^(b)The code **indicates that the males were attracted to the treated group morestrongly than to the control group at a standard of 0.01% (t-test, p =0.0001). ^(c)Acetylated product of sex pheromone of Pseudococcus cryptus^(d)Acetylated product of sex pheromone of Planococcus citriBiological Assay of Attracting Activity Using a Pheromone Trap

Biological assay of sexually attracting activity shown in Table 3 wascarried out as follows. First, a 1-cm² square filter paper, which hadbeen impregnated with each of various attracting substances shown inTable 3 (100 μl of a stock solution in the case of crude sex pheromonesshown in Table 3, and 10 μl of a stock solution in the case ofacetylated products shown therein) and then air-dried, was attached tothe center of a yellow adhesive trap (10 cm height×20 cm width).Thereafter, the trap was placed in a glass chamber with a size of 5m×6.5 m. On the other hand, a filter paper impregnated with only asolvent (100 μl) was used as a control. The three traps were placed in aline at intervals of 1.4 m at a position of a height of 1.8 m. Of these,the trap (control) to which a filter paper impregnated with a solventand then air-dried had been attached was placed in the center. Below thecontrol, a tissue paper on which male pupa at a stage immediately beforeeclosion were attached was placed every day, so that newly emerged adultmales were spontaneously provided from this tissue paper for the test.The traps were exchanged with new traps every morning, so that thenumber of adult males captured by the traps was examined. The resultsare shown in Table 3.

TABLE 3 The number of adult males of Planococcus citri captured byyellow adhesive traps to which various types of attracting substanceswere attached Mean value ± standard error Attracting source (repetition)Acetylated substance 1^(a) 133.3 ± 46.9(4)a^(c) Crude extract of sexpheromone of Planococcus citri 114.8 ± 44.5(4)a Control  0.3 ± 0.3(4)bAcetylated substance 2^(b)  59.4 ± 18.9(5)a Crude extract of sexpheromone of Planococcus citri  84.2 ± 26.2(5)a Control  0.6 ± 0.4(5)b^(a)Acetylated product of sex pheromone of Pseudococcus cryptus^(b)Acetylated product of sex pheromone of Planococcus citri ^(c)Thereare no significant differences among the same characters(Tukey-Kramer-test, p = 0.05).

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention provides a novel ester compound, which has anaction to attract adult males of Pseudococcus cryptus and is useful as asex attractant.

1. Isolated and purified 3-isopropenyl-2,2-dimethylcyclobutylmethyl3-methyl-3-butenoate represented by the following formula (I):


2. A sex attractant comprising, the isolated and purified3-isopropenyl-2,2-dimethylcyclobutylmethyl 3-methyl-3-butenoate; and anorganic solvent selected from the group consisting of pentane, hexane,diethyl ether, acetone, methylene chloride, and combinations thereof. 3.A sex attractant comprising, the isolated and purified3-isopropenyl-2,2-dimethylcyclobutylmethyl 3-methyl-3-butenoateencapsulated in a rubber cap, a capillary, or a plastic capsule.
 4. Asex attractant comprising, the isolated and purified3-isopropenyl-2,2-dimethylcyclobutylmethyl 3-methyl-3-butenoatecontained in a trap.
 5. A sex attractant comprising, the isolated andpurified 3-isopropenyl-2,2-dimethylcyclobutylmethyl 3-methyl-3-butenoatecarried and absorbed on an activated carbon, an inactivated powder or agranule.